PAP increases the phosphorylation of p17 matrix protein.

<p>293 T cells were transfected with the pMenv(-) proviral clone (5 µg) and 0.5 µg 3x-Flag-PAP, 3x-Flag-PAPx, pcDNA3 or MEK_AC vectors. Cells co-transfected with PAP and HIV-1 and cells transfected with HIV-1 alone were additionally treated with 20 µM of the MEK inhibitor PD98059. Particles were purified by ultracentrifugation through a 20% sucrose cushion and equivalent amounts of purified virus (30 ng) were pelleted, lysed and subjected to an endogenous kinase assay using [γ33P-ATP] as a phosphate donor. Samples were resolved on 15% SDS-PAGE and phosphorylated p17 was visualized using a phosphorimager. Fractions of pelleted virus were analyzed via immunoblot using a p17-MA-specific antibody (1∶1,000). Blots are representative of four separate experiments.